The invention relates to a method of preparing a chemically modified hemoglobin solution.
Advances have occurred in recent years in the development of hemoglobin-based blood substitutes. Such transfusional fluids serve as alternatives to whole blood or blood fractions for use as oxygen carriers, plasma expanders, enhancers of radiation and chemotherapy, and scavengers of nitric oxide. Hemoglobin-based blood substitutes are produced by modification of ultra-purified or stroma free hemoglobin using a variety of intermolecular or intramolecular reactions. These various modifications are designed to stabilize the protein, modulate oxygen-binding properties to acceptable physiological levels, and to decrease renal toxicity. Due to the presence of the heme and iron moieties, the hemoglobin molecule has potential toxicity resulting from rapid autooxidation of hemoglobin following free radical interactions and hemoglobin-mediated vasoconstriction. Further, hemoglobin can react with cellular oxidants such as hydrogen peroxide, resulting in the production of both methemoglobin (Fe3+) and ferrylhemoglobin (Fe4+), a strong oxidant reported to cross-link proteins and peroxidize lipids.
Antioxidant systems within the red blood cells control these free radical reactions and protect the function and structural integrity of hemoglobin. For instance, in the red blood cell, endogenous antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), catalyze the breakdown of superoxide and hydrogen peroxide (H2O2), respectively, and hence serve to maintain the functional and structural integrity of hemoglobin. Hence, the oxygen delivery capability of polymerized hemoglobin solutions may be enhanced through the association of these antioxidant enzymes.
One recent experimental approach has resulted in the design of a second generation hemoglobin based oxygen carrier in which the free radical reactivity is limited. The design is based on gluteraldehyde-mediated cross-linking of purified SOD and catalase to purified hemoglobin. See, for example, D""Agnillo et al. (1998) Nature Biotechnol 16:667-671 and U.S. Pat. No. 5,606,025, both of which are herein incorporated by reference. The modification has been reported to prevent H2O2-mediated ferrylhemoglobin formation (D""Agnillo et al. (1998) Free Rad. Bio. Med 24:906-912) and reduce hydroxyl radical generation both in vitro, in a rat hind limb model of ischemia-reperfusion injury (D""Agnillo et al. (1998) Artif. Cells Blood Substit. Immobil. Biotechnol 25:181-192) and in vivo, in a rat model of intestinal ischemia-reperfusion injury (Razack et al. (1997) Artif. Cells Blood Substit. Immobil. Biotechnol. 25:163-180). These studies have led to the proposal that modified hemoglobins with antioxidant activity may reduce the potential toxicity of hemoglobin-based oxygen carriers in certain applications by maintaining the oxygen-carrying ferrous form of hemoglobin and by decreasing the occurrence of potentially harmful hemoprotein-associated free radical species or the release of hemoglobin degradation products which may be harmful to the surrounding tissues.
While the cross-linked polypeptide complex comprising hemoglobin, SOD, and catalase has been suggested to have potential in vivo benefits, the methods taught by U.S. Pat. No. 5,606,025 for the preparation of the modified hemoglobin product comprises combining purified hemoglobin with purified bovine catalase and superoxide dismutase in the cross-linking reaction. There are several practical drawbacks to the addition of enzymes back to purified hemoglobin. First, the addition of bovine or any non-human derived enzymes is potentially a problem based on the immunogenecity associated with the enzymes during human administration. Second, the purification of the enzymes from human blood is costly and may be inhibitory to producing a product economically.
Pyridoxalated hemoglobin polyoxyethylene conjugated (PHP) is a chemically modified, human derived hemoglobin. The PHP molecule is a surface decorated hemoglobin with contains endogenous antioxidant enzyme activities (Privalle et al. (1998) Free Rad. Bio. Med. 25:(Suppl. 1)S65 abstr; Privalle et al. (2000) Acta Physiol. Scand. 167:(Suppl. 645)P200 abstr.; Privalle et al. (1999) Free Rad. Bio. Med. 28:1507-1517 and Talarico et al. (2000) Biochim. Biophys. Acta 1476:53-65; all of which are herein incorporated by reference). PHP, in several in vitro models, has been demonstrated to display a decreased reactivity toward hydrogen peroxide-mediated oxidation when compared to unmodified hemoglobin or xcex1xcex1-crosslinked hemoglobin. Hence, the redox reactivity of the hemoglobin preparations can be manipulated and the presence of endogenous antioxidants may reduce the pro-oxidant potential of hemoglobin. Therefore PHP, by virtue of the presence of endogenous red blood cell antioxidants, may function as a cell free hemoglobin with the antioxidant potential of the intact red blood cell, thus preventing or reducing the pro-oxidant potential of hemoglobin.
A serious problem associated with animal or human derived hemoglobin based blood substitutes arises from the possibility of viral contaminants in the modified hemoglobin solution. Virus elimination and inactivation is commonly performed using either chromatography or heat inactivation. However, treatment of hemoglobin solutions using these methods either removes or abolishes the beneficial activity of the antioxidants associated with the hemoglobin molecule.
For instance, prior methods for the synthesis of a chemically modified hemoglobin product employed filtration means that prevented passage of viral particles but also prevented the passage of endogenous antioxidant enzymes. Alternative methods are provided in U.S. Pat. No. 5,741,894 which discloses a method for the preparation of a pharmaceutical grade hemoglobin. U.S. Pat. No. 5,464,814 discloses a process for preparing a tetramer-free, stroma-free, cross-linked, polymerized, pyridoxylated hemoglobin. Both methods employ a filtration step that allows the passage of hemoglobin, endogenous antioxidant enzymes, and viral particles. The viral particles are subsequently inactivated in a heat treatment step that abolishes the activity of both the viral particles and antioxidant enzymes. In other methods, the viral particles are removed along with the antioxidant enzymes by chromatography. See, for example, U.S. Pat. Nos. 5,264,565 and 5,988,361.
Alternatively, U.S. Pat. No. 5,194,590 discloses a method of preparing a modified hemoglobin comprising the lysis of red blood cells and removal of red blood cell stroma by a microporous filtration process that allows hemoglobin and the enzymatic contents of the red blood cells to pass through the filter using a 0.1 xcexcm filter. Following chemical modification, a three step purification process is performed which removes unmodified tetramers and also reduces viral contamination. The additional chromatography step is essential to remove the viral contaminants from the hemoglobin solution, which passed through the 0.1 xcexcm filter.
Because the antioxidant enzymes provide advantageous properties to the modified hemoglobin, methods are needed to prepare purified hemoglobin that is viral free yet still retains antioxidant activity.
The present invention provides methods and compositions for the separation of hemoglobin molecules and endogenous antioxidant enzymes from red blood cell stroma. The method of separation further reduces the level of potential adventious agents, such that a substanitally viral free hemoglobin/antioxidant solution is produced. Specifically, the present invention provides a method for preparing a modified hemoglobin solution comprising endogenous antioxidant enzymes of the red blood cells. The method comprises contacting a stroma free hemoglobin solution with at least one filtration means that retains viral particles and allows passage of a filtrate comprising hemoglobin and endogenous antioxidant enzymes, chemically modifying the filtrate with an agent, and isolating the modified hemoglobin and antioxidant enzymes. In one embodiment, the method of the present invention is used to synthesize pyridoxalated hemoglobin polyoxyethylene conjugate (PHP).
The present invention also provides a method of preparing a chemically modified hemoglobin. The method consists of contacting a stroma free hemoglobin solution with at least one filtration means, wherein a first filtration means retains viral particles and allows passage of a filtrate comprising hemoglobin and endogenous antioxidant enzymes and the filtrate is substantially free of viral contamination; chemically modifying the filtrate with an agent; and, isolating the modified hemoglobin and antioxidant enzymes.
The present invention further provides a composition comprising a modified hemoglobin solution. The modified hemoglobin solution comprises chemically-modified hemoglobin molecules and at least one endogenous antioxidant enzyme wherein the chemical modification comprises crosslinking with bifunctional activated polyoxyethylene (POE). The endogenous antioxidants present in the solution retain enzymatic activity, and the solution is substantially free of viral contamination.